Seven transmembrane-spanning receptors (7TMRs or G protein-coupled receptors, GPCRs) represent the largest family of signal-transducing molecules known. 7TMRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of 7TMRs has been found in a growing number of human diseases and 7TMRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how 7TMRs function is an important goal of biological research. We have used TRH receptors (TRH-R) and TSH receptors (TSH-R) as model 7TMRs to study their structure and function. During this year, we studied several new aspects of the structure and function of TSH-Rs. 1) An important observation for TSH-Rs is that they are expressed on cells other than the thyrocyte but the role(s) of TSH-R in these cells is not well understood. One prominent cell that expresses TSH-Rs is the pre-osteoblast that is the precursor for forming new bone. Also, it has been known for a number of years that TSH-R can activate several different signal transduction pathways in model cell systems such as HEK and CHO cells. However, whether these pathways are important in TSH-R signaling that leads to differentiation of cells that express TSH-R in vivo was not known. We have extended our previous observations of the effect of TSH on differentiation of human pre-osteoblasts to normal human thyrocytes. We showed that TSH-R mediates differentiation of human thyrocytes but not proliferation (as had been previously thought) and that different signaling pathways regulate up-regulation of thyroid-specific genes. These data show that the human thyrocyte responds to TSH differently than the more commonly studied rodent and dog thyrocytes and are important because it is now apparent that non-human thyrocytes are not good models of human cells. We also showed that there is an important cross-talk between TSH-R and the receptor for insulin-like growth factor-1 (IGF-1R) and that drugs to increase differentiation would likely need to activate both TSH-R- and IGF-1R-initiated pathways. 2) Graves' ophthalmopathy (GO) is a troublesome component of Graves' disease (GD) that occurs in 25% of GD patients but for which there is no medical therapy. We study GO in cells in primary culture taken from the retro-orbital space of patients with GO at decompression surgery. We use these cells to study GO as there is no well-developed animal model of GO. We found a critical interaction between the TSH-R and the IGF-1R in these cells also. We showed that this interaction (cross talk) allowed for a synergistic increase in the production of hyaluronan (hyaluronic acid, HA), which is a major component of the dysregulated extracellular matrix in GO. We have now shown that this cross-talk is similar in mechanism to other interactions between GPCRs and receptor tyrosine kinases. It was proposed by others that there may be immunoglobulins (auto-antibodies; GO-Igs) in the blood of GO patients that can bind to and activate TSH-Rs and IGF-1Rs. We have now provided new evidence that there are no auto-antibodies that directly bind to and activate IGF-1R signaling. Nevertheless, we showed that inhibition of IGF-1R signaling may have a beneficial role in GO treatment. (This idea was recently confirmed in a study in patients with GO.) Our observations allow for the definitive conclusion that IGF-1R activation by GO-Igs occurs via TSHR/IGF-1R cross talk rather than direct binding to IGF-1R, and this cross talk is important in the pathogenesis of GO.